Ideally, an immunogen composition should potentiate long-lasting expression of functionally active antibodies, elicit cell-mediated immunity (“CMI”), and enhance production of memory T- and B-lymphocytes with highly specific immunoreactivity against an invading antigen. In addition to providing a defense upon immediate challenge with a foreign antigen, these responses should provide protection against any future encounters of the host with a specific antigen.
Situations in which it is desirable to elicit these types of sustained responses include the development of protective immunity against infectious agents or their products, against tumor antigens for the treatment of cancer and as a form of sterilization or birth control in which an immune response is induced against components of the mammalian reproductive system such as human chroionic gonadotrophin (HCG). The complexity of the immune system in mammals is well established and many factors contribute to the type of immune response that occurs when a foreign substance is encountered. Three outcomes are possible: it may be ignored, it may induce a state of unresponsiveness or tolerance such that a future encounter with that antigen would not result in an immune response or it may elicit an immune response the quality of which is influenced by the many factors. These include the form of the antigen, whether soluble or particulate in nature, the foreignness of the antigen, i.e., how far removed the antigen is from the host on the phylogenetic tree, the stability of the antigen to degradative enzymes of the host and the ability of the antigen to persist in the host for long periods of time. It can be appreciated that the immunogenicity of an antigen that elicits a weak immune response may be improved by manipulation of one or more of these parameters.
Traditionally the immunogenicity of an antigen has been improved by injecting it in a formulation that includes an adjuvant. Adjuvants non-specifically augment immune responses and their ability to potentiate immune responses has long been recognized. A wide variety of substances, both biological and synthetic, have been used as adjuvants in experimental systems. These include mycobacteria, oil emulsions, liposomes, polymer microparticles and mineral gels. The mechanism by which adjuvants enhance immune responses is not uniform but their effects may include retention of antigen at the site of administration such that the antigen is released to the body slowly over time or array of the antigen in a particulate form so that it is more easily recognized by lymphocytes and taken up by antigen presenting cells. Adjuvants consisting of microbial products generally act by enhancing the uptake of antigens by professional antigen-presenting cells and/or by stimulation of the innate immune system, which in turn leads to more potent stimulation of lymphocytes themselves.
For therapeutic use in humans, however, the toxic side effects of many adjuvants used as research tools have limited their use. Currently only aluminum salts are approved for use in humans in the United States and these are a component of many common vaccines, e.g., tetanus and DTP. However, there is some concern regarding the safety of aluminum salts (Malakoff, “Aluminum is Put on Trial as a Vaccine Booster,” Science, 2000, 288, 1323). When aluminum salts are used as an adjuvant, the antigen is adsorbed to the aluminum salt, thereby arraying the antigen in particulate form as well as forming a depot of antigen, which is released slowly over time. Even in this formulation, however, vaccines are usually administered several times over a time span of months in order to elicit an immune response that can confer protection on the host upon subsequent encounter with the antigen, e.g., microbe, itself. Thus although vaccines for a variety of infectious diseases are currently available, many of these, including those for tetanus and hepatitis B, require more than one administration to confer protective benefit. These limitations are extremely problematic in countries where healthcare is not readily available or accessible. Moreover, compliance is also a problem in developed countries, particularly for childhood immunization programs. For example, a child in the United States may be scheduled to receive a total of 16 vaccine injections by age 18 months and 35 vaccine injections by age five.
Research efforts into improving vaccines have developed along many different but parallel courses but of great importance have been the development of new compositions and delivery systems, which could reduce the number of injections, required but still elicit long-lasting protective immunity. Included in this are the development of new and novel adjuvants with improved safety profiles. In research efforts to reduce the immunization regimens research has been directed towards both development of single dose delivery vehicles and development of non-injectable vaccines.
Mucosal vaccine strategies have recently emerged as an attractive potential alternative to injectable vaccines. Mucosal administration would have many potentially desirable attributes. This form of administration is relatively easy and low cost, especially when compared to injection regimens. As such, mucosal administration has significant potential to improve compliance in both developing and developed countries relative to vaccine injections, particularly for childhood immunization programs. Another advantage of mucosal administration compared to injection is a reduced risk of contamination with elimination of the use of needles.
Perhaps the most compelling reason for developing mucosal vaccine delivery techniques, however, is development of a first line of immunity defense, by generating local immunity at the mucosal site of entry for many invading pathogens. Moreover some investigators have reported that a common mucosal immune system exists, whereby mucosal immunity induced at one site can lead to immunity at a distal mucosal site (McGhee, J. R. et al. The mucosal immune system: from fundamental concepts to vaccine development. Vaccine 1992, 10:75-88). This suggests that significant benefits can be achieved by the delivering of vaccines in a non-invasive way, e.g. intranasally or other mucosal route, to elicit immunity to a wide range of pathogens that may enter at different mucosal sites, e.g. HIV, HPV. In addition, delivery of an antigen via a mucosal site has the potential to generate a systemic immune response as well.
A mucosal immune response consists of all the components of the systemic immune system including the ability to generate cell-mediated and humoral responses. Cells of the immune system are distributed throughout mucosal tissues and include T and B cells and cells capable of antigen presentation, such as dendritic cells and monocytes/macrophages (Neutra, M. R. et al. Antigen sampling across epithelial barriers and induction of mucosal immune responses. 1996. Ann. Rev. Immunol. 14: 275-300). The main antibody class present in the mucosal immune system is IgA, which is exported in polymeric form into mucosal secretions. Once in the lumen, IgA antibodies prevent attachment of infectious agents or their toxins to mucosal epithelia thereby providing a first line of defense against infection. Detection of IgA antibodies in washes of mucosal surfaces indicates the generation of a mucosal immune response to an antigen.
Although there is great promise for mucosal administration of vaccines, delivery of many antigens, such as proteins and peptides, to the mucosal tissue does not necessarily result in the generation of an immune response. The generation of mucosal immunity to antigens is dependent upon the same criteria as is the systemic immune response, namely that the antigen must be presented appropriately in a form that will lead to stimulation of T and B lymphocytes. This often means that an adjuvant is required to non-specifically enhance the mucosal immune response as well as the systemic response. In addition to this requirement, uptake of antigens from the mucosae requires that the antigen is able to penetrate the epithelial barrier and gain access to the underlying lymphoid tissue. For these reasons mucosal delivery of antigens may result in low bioavailability and also may induce immunological tolerance (e.g. Lowrey, J. L. et al. Induction of tolerance via the respiratory mucosa. Int. Arch. Allergy Immunol. 1998, 116: 93-102).
In recent years many adjuvants and delivery systems have been evaluated for their ability to enhance the immune response to mucosally administered antigens. These include bacterially-derived products such as monophosphoryl lipid A (Baldridge, J. R. et al. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. 2000. Vaccine 18: 2416-2425), immunostimulatory DNA sequences (Homer, A. A. et al. Immunostimulatory DNA is a potent mucosal adjuvant. 1998. Cell. Immunol. 190: 77-82; McCluskie, M. J. et al. Intranasal immunization of mice with CpG DNA induces strong systemic and mucosal responses that are influenced by other mucosal adjuvants and antigen distribution. 2000. Mol. Med. 6:867-877), outer membrane proteins of Neiserria meningitidis serogroup B (Levi, R. et al. 1995. Intranasal immunization of mice against influenza with synthetic peptides anchored to proteosomes. Vaccine 13: 1353-9), and bacterial toxins such as cholera toxin (CT) subunit B and E. coli enterotoxin (ET) (Isaka, M. et al. Systemic and mucosal immune responses of mice to aluminum-adsorbed or aluminum-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit. 1998, Vaccine 16: 1620-1626; Holmgren, J. et al. Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems.1993 Vaccine 11: 1179-1184; Tamura, S. et al. Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine 1994, Vaccine 12: 419-426; Goto, N. et al. Safety evaluation of recombinant cholera toxin B subunit produced by Bacillus brevis as a mucosal adjuvant. 2000. Vaccine 18: 2164-2171; and Barchfeld, G. L. et al. The adjuvants MF59 and LT-K63 enhance the mucosal and systemic immunogenicity of subunit influenza vaccine administered intranasally in mice. 1999. Vaccine 17: 695-704). However, the inherent toxicity of bacterial toxins generally precludes their use in human vaccines. Detoxified mutants of both CT and ET have been produced (Pizza, M., et al. A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit. 1994. J Exp Med 180, 2147-53; Yamamoto, S., et al. A nontoxic mutant of cholera toxin elicits Th2-type responses for enhanced mucosal immunity. 1997. Proceedings of the National Academy of sciences of the United States of America 94, 5267-72) but these are generally less effective than the wild type toxins. Therefore, the development of novel mucosal vaccine delivery systems that do not induce systemic side effects or damage the mucosal membrane is of prime importance.
A substantial research effort has also been devoted to the improvement of vaccine delivery systems for injectable formulations (see Edelman, R. in “Vaccine Adjuvants” ed. D. T. O'Hagan, Humana Press, Totowa, N.J., 2000). These include microparticles, bacterial products, slow release polymers and other vehicles.
One product in which there has been a lot of recent interest for both mucosal delivery of vaccines and drugs as well as for use as a systemic adjuvant is chitosan, a cationic biopolymer derived from deacetylated chitin. Chitosan has been shown to act as a penetration enhancer to the extent its presence appears to improve the uptake of at least some drugs through the nasal mucosa. The mechanism of action is not completely understood but is thought to be due to opening of the tight junctions between cells in the nasal epithelium as well as increasing residence time of the drug within the nasal passages (Illum, L. et al. Chitosan as a novel nasal delivery system for peptide drugs. 1994. Pharmaceutical Research 11: 1186-1189). Chitosan formulated with other excipients such as lysophosphatidylcholine has also been shown to further enhance uptake of proteins across epithelia (Witschi, C. and R. J. Mrsny. In vitro evaluation of microparticles and polymer gels for use as nasal platforms for protein delivery. 1999, Pharmaceutical Research. 16: 382-390). In addition, chitosan has been shown to have pro-inflammatory activity, activating macrophages and stimulating secretion of pro-inflammatory cytokines such as TNFα and IL1β from monocytes in vitro. Therefore it appears to act as an immunological adjuvant in at least some circumstances. These properties of chitosan have been exploited in the development of vaccines for both intransal and systemic (e.g. intraperitoneal) delivery (McNeela, E. A. et al. 2001. A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM197) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery. Vaccine 19: 1188-1198; Bacon, A. et al. Carbohydrate biopolymers enhance antibody responses to mucosally delivered antigens. Infection and Immunity 2000 68: 5764-5770; Jabbal-Gill, I. et al. Stimulation of mucosal and systemic antibody response against Bordatella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice. Vaccine 16: 2039-2046; and Seferian, P. G. and M. L. Martinez. Immune stimulating activity of two new chitosan containing adjuvant formulations. Vaccine. 2001, 19: 661-668.). However, reports have commented on the intragroup variation occurring when chitosan is used systemically (Jabbal-Gill, I. et al. Vaccine 16: 2039-2046) and others have found that further formulation of antigen and chitosan within an emulsion raises more potent antibody responses than a mixture of antigen/chitosan alone (Seferian, P. G. and M. L. Martinez. Vaccine 2001. 19: 661-668.).
Although improvements have been made in the area of vaccines there is still a strong need to develop immunogen formulations that reduce or eliminate the need for a prolonged injection regimen. There is also a need to develop immunogen formulations that are well suited for mucosal delivery and that are effective for providing mucosal as well as systemic immunity. There is a further need for immunogen formulations that enhance mucosal immunity locally and systemically with no or reduced side effects and that are administrable without altering the integrity of the mucosal membrane.